1. Field of the Invention
The present invention relates to a reagent kit for determining a characteristic of a tissue based on expression level and activity value obtained by measuring a cyclin-dependant kinase in the tissue sampled from a patient.
2. Description of the Related Arts
As the cell-cycle regulating factor governing cell proliferation of organisms, cyclin-dependant kinase (hereinafter, abbreviated as CDK) that is a positive regulating factor is present. Upon being exposed to activation such as phosphorylation or the like, CDK binds to cyclin resulting in a complex of CDK and cyclin (hereinafter, abbreviated as active type CDK in some cases). CDK is thus activated. In this way, CDK exhibits activity at particular stage of cell cycle according to the type thereof. Further, it has been known that cyclin-dependant kinase inhibitor (hereinafter, abbreviated as CDK inhibitor) that is a negative regulating factor binds to CDK and/or cyclin-CDK complex thereby inhibiting activity value of CDK.
Data of the expression level and the activity value of cell-cycle regulating factors may be used as convenient indexes for determining a characteristic of a tissue sampled from a patient such as type and malignancy of disorders associated to controls of cell cycle. For example, it is known that although expression of CDK in cells is normally constant, its expression becomes high in cells where proliferation is induced by a growth factor. However, as mentioned above, mechanism of activation of CDK is complicated one in which CDK, cyclin and CDK inhibitor are closely related and therefore, mere data of expression level of CDK, cyclin and CDK inhibitor, or mere data of activity value of CDK are not enough to be used as the indexes for determination of a characteristic of a tissue.
As used herein, “Expression level” denotes a target protein amount (units corresponding to number of molecules) obtained by measurement using tissue solution prepared from the tissue. Expression level can be determined by the conventional known method for determining amount of target protein from a protein mixture. For example, expression level can be determined using ELISA, western blotting, the method disclosed by U.S. Patent Application Publication No. 2004-0214180 or the like. A target protein can be trapped by using a specific antibody. In trapping of CDK1, for example, all of CDK1 (including CDK1 alone, complex of CDK1 and cyclin and/or CDK inhibitor, complex of CDK1 and other compounds) existing in said tissue solution can be trapped by using an anti-CDK1 antibody.
Further, CDK activity value denotes kinase activity level showing how many substrates (e.g., histone H1 for activated CDK1 and activated CDK2, Rb (Retinoblastoma protein) for activated CDK4 and activated CDK6) can be phosphorylated while CDK is bound to a specific cyclin, and is expressed by U (unit). In other words, it is possible to determine the activity value of CDK1 or CDK2 by checking how much histone H1 are phosphorylated by activated CDK1 or CDK2. Further, it is possible to determine the activity value of CDK4 or CDK6 by checking how much Rb (Retinoblastoma protein) is phosphorylated by activated CDK4 or CDK6. The activity value of CDK can be determined by conventional enzyme activity measurement method that uses radioactive materials as a label. Specifically, a method using 32P-labeled adenosine5′-O-(3-triphosphate) (γ-[32P]-ATP) is mentioned. With this method, monophosphoric group derived from 32P-labeled ATP is taken into a substrate by CDK action, and amount of 32P taken into a substrate is detected by autoradiography or scintillation counter to determine the amount of substrate being phosphorylated, and activity value of CDK is calculated from the amount of the substrate. Besides, U.S. Patent Application Publication No. 2002-01647673 discloses a measuring method without using radioactive materials as the label. Specifically, with this method, adenosine5′-O-(3-thiotriphosphate) (ATP-γS) and a substrate are reacted, labeling material is bound to sulfur atom of mono-thiophosphoric acid group being taken into the substrate to determine the amount of labeling. For example, when a fluorescent material is used as the labeling material, fluorescent amount is measured as the amount of labeling.
At present, determination of a characteristic of a tissue sampled from a patient plays an important role at clinical scenes in terms of diagnosis of malignancy of cancers (easily develops metastasis, liable to recurrence, poor prognosis). For example, diagnosis of cancers is performed using information made available from measurements of tumor marker, tissue diagnosis and cytological diagnosis by biopsy, TNM classification (T: Size of primary focus, N: Level of lymph node metastasis, M: Distant metastasis), measurements of DNA content using fluorescent display type cell sorter or the like. However, the following problems are involved in each of the methods:
Diagnosis sensitivity is low.
Diagnosis is difficult since final judgment is made based on observer's own experiences.
Accurate forecasting of prognosis is not available though pathological condition is grasped at diagnosis.
Skilled technology is required.
These problems are directly reflected on the difference of judgment by individuals and of judgment by medical institutions in the diagnosis of malignancy of cancers, thereby resulting in a factor for scattering of diagnosis of malignancy of cancers.
In a case where therapeutic strategy of cancer should be established, checking of sensitivity of cells in the tissue to drugs such as anticancer agents is so important as well as diagnosis of malignancy. For methods to be used on such an occasion, anticancer agent sensitivity test for tumor cells performed in vitro is available, and MTT (3-(4,5 dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) assay and DISC (Differential Staining Cytotoxicity) method are known. However, positive prediction rate of either of these methods is less than 70% which is insufficient to be used as the index to know whether or not anticancer agent treatment should be initiated (Weisenthal, L. M. and Nygren, P. (2002) Current status of cell culture drug resistance testing (CCDRT), http://weisenthal.org/). As mentioned above, measurement of expression level of CDK in a tissue sampled from a patient is disclosed by U.S. Patent Application Publication No. 2004-0214180, and measurement of activity value of CDK is disclosed by U.S. Patent Application Publication No. 2002-0164673, a kit for determining a characteristic of a tissue based on the expression level and the activity value of CDK has not been developed yet. Each of measurements of expression level of CDK and activity value of CDK involves a number of reagents and reagents with different storage conditions are mixed, which indicates that handling of these reagents is significantly cumbersome.